The Live Attenuated Influenza Vaccine (LAIV) has been shown to have greater efficacy in children than its inactivated counterpart. However, due to an unacceptable safety profile, it is not licensed in either asthmatics or children under the ag of two. If one could further attenuate LAIV while retaining immunogenicity, these safety concerns could be alleviated and LAIV usage could be extended to cover these groups. We recently discovered a novel influenza virus mutant that has increased temperature sensitivity, compared to conventional LAIV. The genome of this virus (designated SGR-ts) is characterized by five point mutations within the viral polymerase, when compared to a phenotypic revertant virus with normal temperature sensitivity. Three of these mutations (in PB1 and PB2) are sufficient to almost completely eliminate viral polymerase activity at 37oC, while having no effect on activity at 34oC; importantly, none of these mutations is shared with the current LAIV. These findings suggest that it should be possible to increase the safety of LAIV, by reducing its shutoff temperature (the temperature at which polymerase activity is abolished) to 37oC. Our central hypothesis is that the safety and effectiveness of LAIV can be increased by: (1) using newly discovered polymerase mutations to decrease the shutoff temperature for viral replication (increasing safety) and (2) using selective codon optimization to increase expression of the major virus antigen, hemagglutinin [HA] (increasing immunogenicity). Aim 1 will focus on the construction and in vitro characterization of an enhanced LAIV. To do this, we will identify the minimal essential mutations necessary for the ts polymerase phenotype of SGR-ts, using viral replication assays at selected temperatures (33, 37, and 39oC). We will test whether introduction of these mutations into a conventional H1N1 LAIV can further reduce the viral shutoff temperature, while preserving replication at 33oC - thus creating a ts enhanced H1N1 LAIV. Finally, we will confirm the reproducibility of our findings in the context of an H3N2 LAIV, and we will test whether the ts-enhanced H1N1 LAIV can be further improved by using a codon optimization strategy to increase HA expression. Aim 2 will compare the safety and efficacy of a ts-enhanced H1N1 LAIV (+/- a codon optimized HA) to that of a conventional H1N1 LAIV (also +/- a codon optimized HA). Safety will be assessed by determining MLD50 in mice, as well as weight loss over the course of infection, and virus titers in lung and nasal tract following high-dose infection. Immunogenicity and protective efficacy will be assessed by measuring titers of HA-binding and virus-neutralizing antibodies in immunized mice, and by testing the protection of immunized mice from a lethal H1N1 virus challenge. Finally, we will examine the safety and efficacy of our LAIVs in a second animal model - ferrets - since this model was used during studies that led to the original licensure of LAIV.